Breast cancer is the most common cancer in women, aside from skin cancer. In 2006, according to the National Cancer Institute, approximately 41,000 women per year in the United States die from the disease. Based on current rates, 13.2% of women born today will be diagnosed with breast cancer at some time in their lives. Intensive research has led to advances in diagnosis and treatment; however, serious problems still exist, including low cure rates, substantial adverse effects and resistance to certain therapies. Given that breast cancer is a group of diseases, each having distinct molecular properties, molecularly targeted drugs have emerged as important anti-cancer therapeutics in recent years.
In 25-30% of breast cancers, amplification and overexpression of the growth factor receptor gene HER2 (human epidermal growth factor receptor-2, also known as neu/erbB2) is associated with enhanced tumor aggressiveness and a high risk of relapse and death (Slamon, D., et al., 1987, Science 235:177; Yarden, Y., 2001, Oncology 1:1). This oncogene encodes a 185 kilodalton (kDa) transmembrane receptor tyrosine kinase. As one of the four members of the human epidermal growth factor receptor (EGFR) family, HER2 distinguishes itself in several ways. First, HER2 is an orphan receptor. No high-affinity ligand has been identified. Second, HER2 is a preferred partner for other EGFR family members (HER1/EGFR, HER3, and HER4) for the formation of heterodimers, which show high ligand affinity and superior signaling activity. Third, full-length HER2 undergoes proteolytic cleavage, releasing a soluble extracellular domain (ECD). Shedding of the ECD has been shown to represent an alternative activation mechanism of full-length HER2 both in vitro and in vivo, as it leaves a membrane-anchored fragment with kinase activity. The central role of HER2 in EGFR family signaling correlates with its involvement in the oncogenesis of several types of cancers, such as breast, ovarian, colon, and gastric cancers, regardless of its expression level (Slamon, D., et al., 1989, Science 244:707; Hynes, N., et al., 1994, Biochem. Biophys. Acta. 1198:165). HER2 may also render tumor cells resistant to certain chemotherapeutics (Pegram, M., et al., 1997, Oncogene 15:537). Given its vital role in tumorigenesis, HER2 is an important target for cancer therapeutics.
As a cell membrane receptor, HER2 is composed of an extracellular domain (ECD) (632 amino acids), a transmembrane domain (22 amino acids), and an intracellular domain with tyrosine kinase activity (580 amino acids). As initially transcribed, the pre-mRNA for HER2 contains 27 exons and 26 introns. The fully spliced HER2 mRNA from which the introns have been spliced out is composed of 27 exons. Upon expression, HER2 protein is translocated to the cell surface. Activated through constitutive homo-dimerization and ligand-stimulated hetero-dimerization, HER2 protein directs subsequent steps in signal transduction, which affect cell growth, survival, and differentiation.
HER2 has been validated as a therapeutic target for several epithelial malignancies, including those originating in the breast, lung and colon. Currently there is only one FDA-approved therapeutic for HER2 positive breast cancer, Herceptin® (Colomer, R., et al., 2001, Cancer Investigation 19:49). Herceptin is a recombinant humanized monoclonal antibody that selectively binds to the HER2 extracellular domain with high affinity (Kd=5 nM). Alone or in combination with chemotherapy, Herceptin has been shown to inhibit the proliferation of human tumor cells that overexpress HER2 (Slamon, D., et al., 2001, N. Engl. J. Med. 344:783; Baselga, J., et al., 1998, Cancer Research 58:2825).
However, this antibody-based therapeutic reagent has certain limitations. First, its inhibitory effect is restricted to the HER2 displayed on the cell surface; intracellular HER2 molecules are still available for mitogenic signaling. Second, Herceptin can be bound and thus “neutralized” by circulating ECDs that are released by proteolysis of membrane-bound HER2 (Brodowicz, T., et al., 1997, Int. J. Cancer 73:875). Finally, as with many other drugs, prolonged treatment with Herceptin leads to acquired resistance (Kute, T., et al., 2004, Cytometry Part A 57A:86). Another anti-HER2 antibody, pertuzumab, has been shown in a phase II clinical trial to have activity in ovarian cancer (Gordon, M. S., et al., 2006, J. Clin. Oncol. 24:4324).
At least two autoinhibitors of HER2, translated from alternatively spliced HER2 mRNA species, have been reported. These are HER2-68 and HER2-100. Retention of intron 8 in the HER2 mRNA produces a variant mRNA that encodes a 68-kDa HER2 protein, HER2-68 or Herstatin. Retention of Intron 15 produces a variant mRNA that encodes a 100-kDa truncated HER2 protein, HER2-100. Both HER2 splice variants are soluble and act as dominant-negative inhibitors of HER2, most likely through interfering with receptor dimerization.
When HER2-100 is overexpressed in MCF-7 breast cancer cells, spontaneous proliferation and heregulin-mediated soft agar colony formation of MCF-7 cells decreases (Aigner, et al., 2001, Oncogene, 20(17):2101). Downstream signaling pathways are also negatively affected.
The 68-kDa variant, or Herstatin, has been characterized in more detail. Upon expression in tumor cells, Herstatin is secreted and binds to HER2-presenting cells with high affinity (Kd=14 nM); Herstatin also binds to HER1 and HER4. Herstatin interferes with the activity of HER2 and other EGFR family members, and thus interferes with their downstream signal transduction. Herstatin has been reported to cause tumor growth arrest and inhibition of breast cancer cell growth. Herstatin overcomes tamoxifen resistance in HER2 positive breast cancer cells (Justman, Q., et al., 2003, J. Biol. Chem. 277:20618; Jhabvala-Romero, F., et al., 2003, Oncogene 22:8178). Therefore, Herstatin has been recognized as a promising anti-cancer drug candidate (Stix, G., 2006, Scientific American 294:60). With both HER2-100 and Herstatin, a progressive loss of their expression in more advanced tumors has been observed.
HER3 (human epidermal growth factor receptor-3, erbB3) is a receptor protein that plays an important role in regulating normal cell growth. HER3 lacks an intrinsic kinase activity and relies on the presence of HER2 to transduce signals across the cell membrane. As initially transcribed, the pre-mRNA for HER3 contains 28 exons and 27 introns. The fully spliced HER3 mRNA from which the introns have been spliced out is composed of 28 exons.
Two natural splice variants of HER3, p45 and p85, have been reported. Both are soluble, secreted, truncated proteins generated through alternative splicing of HER3 pre-mRNA. The mRNAs that code for each of these splice variants do not allow translation of the full-length HER3 protein, and instead generate truncated proteins. In particular, the p85 form results from the retention of Intron 13 (FIG. 12). These proteins block Heregulin-stimulated activation of HER3, HER2 and HER4, thereby inhibiting the growth of cells through the EGFR signaling pathway. Using a dominant negative truncated form of HER3 to inhibit HER2/HER3 signaling, it is possible to protect against pulmonary fibrosis (Nethery, D. E., et al., 2005, J. Appl. Physiol. 99:298).